The long-term objective of this work is to develop an experimental basis for gene therapy of hemophilia B, the bleeding diathesis that results from an absence of coagulation Factor IX. Previous work with retroviral vectors expressing F.IX has achieved long-term expression, but levels of expression have been very low. More recent work making use of adenoviral (Ad) vectors has been successful at achieving high plasma levels of F.IX, but expression has been transient, and the level of F.IX fails to rise following repeat administration of the vector. In this proposal, we will 1) make modifications in the F.IX cassette to allow optimal expression of F.IX from viral vectors. We will also make use of novel strategies aimed at achieving long-term expression from 2) adenoviral vectors, including 3) use of an engineered Ad vector that expresses lower levels of viral proteins, and results in more sustained expression in transduced animals than first generation Ad vectors; 4) use of in utero gene transfer techniques, that may allow induction of tolerance to adenoviral proteins, and achieve sustained expression on that basis; and 5) use of an immunoisolation device to create an immunoprivileged site that may permit prolonged expression from transduced cells contained within the device. We have recently been successful at generating an AAV-F.lX vector that we have used to transduce a human hepatoma cell line. We propose to exploit this promising result by 6) determining whether an AAV-F.lX vector can direct long-term expression of F.IX in transduced cells. Ad-F.IX vectors will be tested in cell culture, and in animal models including mice, rats, and canines with hemophilia B. Published experience with AAV as a tranducing vector is much more limited than that with Ad vectors. The initial work with AAV-F.lX will be in cell culture; if results warrant, we will proceed to work in an animal model.